This is a K01 application from NIDDK in the field of Clostridium difficile infection (CDI). My long- term career objective is to utilize knowledge acquired through my work and others to develop measures of prevention and therapy against CDI and other enteric pathogens-associated diseases. The object of this study is to elucidate the signaling pathway of C. difficile toxin- mediated TNF- production and develop a novel therapy against CDI by targeted blocking TNF- production. Three specific aims will be pursued. Specific Aim 1: Identify subsets of immune cells that are major producer of TNF- in response to C. difficile toxins in vivo, based on our hypothesis that intestinal dendritic cells (DCs) and macrophages are major TNF- producer during the bacterial infection. Specific Aim 2: Investigate the signaling events that lead to toxin-mediated TNF- production in macrophages. Specific Aim 3: Evaluate blocking TNF- production as an adjunctive therapy against intestinal inflammation in both primary and recurrent CDI. For specific aim1, TNF- producing immune cells will be identified by immuofluorescence staining and immunohistochemistry in mouse ileal loop model and C. difficile infection in mice. To more precisely assess the roles of DCs and macrophages in TNF- production, DC- or macrophage- depleted mice will be used. For specific aim 2, multiple approaches including siRNA knockdown, Western-blot analysis, RT-PCR will be performed to identify the involvement of small Rho GTPases and dual specificity phosphatases (DUSPs) in C. difficile toxin-induced TNF- production. In specific aim 3, a novel glucan particle (GP)- dependent siRNA delivery system specifically targeting macrophages and DCs will be employed to evaluate blocking TNF- production as an adjunctive therapy against intestinal inflammation in CDI. PUBLIC HEALTH RELEVANCE: This K01 project is designed to find out novel therapeutic targets and to test a novel treatment approach to reduce severity of inflammation in a mouse model of primary and recurrent Clostridium difficile infection.